Mutations in the nucleotide binding domain of the alpha subunits of the F1-ATPase from thermophilic Bacillus PS3 that affect cross-talk between nucleotide binding sites.
Inactivation of MF1 (bovine mitochondrial F1-ATPase) with 5'-p-fluorosulfonylbenzoylethenoadenosine is caused by labeling alpha Y244 [Verburg, J. G., and Allison, W. S. (1990) J. Biol. Chem. 265, 8065-8074]. In the crystal structure [Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], alpha Y244 is hydrogen bonded to alpha R304 which is also hydrogen bonded to alpha Y300. The catalytic properties of mutant alpha 3 beta 3 gamma subcomplexes of the TF1-ATPase from the thermophilic Bacillus PS3 containing the alpha F244C, alpha R304C, and alpha Y300C substitutions have been examined. Each has unique features for hydrolyzing ATP and forming inhibitory ADP-fluoroaluminate complexes in catalytic sites. Unlike wild-type, the (alpha R304C)3 beta 3 gamma and (alpha Y300C)3 beta 3 gamma subcomplexes entrap inhibitory MgADP in a catalytic site during turnover which fails to dissociate when ATP binds to noncatalytic sites. Although the hydrolytic properties of the (alpha F244C)3 beta 3 gamma subcomplex and wild-type are similar, the mutant forms ADP-fluoroaluminate complexes 7 times faster than wild-type when Al3+ and F- are added to it in the presence of excess ADP and Mg2+. It also resists inhibition by high Mg2+ concentrations in the assay medium. At least one noncatalytic site of the (alpha F244C)3 beta 3 gamma subcomplex has increased affinity for ADP, indicating that the enhanced rate of formation of the ADP-fluoroaluminate complex reflects augmented cooperativity between noncatalytic and catalytic sites. The rate of formation of the ADP-fluoroaluminate complex in (alpha Y300C)3 beta 3 gamma increases only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type. When Al3+ and F- are added to the (alpha Y300C)3 beta 3 gamma subcomplex after incubation with excess ADP and Mg2+, ADP-fluoroaluminate complexes are formed in three catalytic sites rather than two observed with the other subcomplexes. Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that alpha F244, alpha R304, and alpha Y300 of TF1 are part of a pathway that propagates conformational signals from one catalytic site to another.
Grodsky
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Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla 92093-0601, USA.
Dou
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C
Allison
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WS
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000535:Aluminum; D001120:Arginine / Q000235:genetics; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D005461:Fluorine; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; C053629:fluoroaluminum; D005461:Fluorine; D014443:Tyrosine; D010649:Phenylalanine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D000535:Aluminum; D006180:Proton-Translocating ATPases; D003545:Cysteine